bioRxiv preprint doi: https://doi.org/10.1101/225581; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota
2
3 Lesley Hoyles1*, Maria L. Jiménez-Pranteda2*, Julien Chilloux1*, Francois Brial3, Antonis
4 Myridakis1, Thomas Aranias3, Christophe Magnan4, Glenn R. Gibson2, Jeremy D. Sanderson5, Jeremy
5 K. Nicholson1, Dominique Gauguier1,3, Anne L. McCartney2† and Marc-Emmanuel Dumas1†
6
7 1Integrative Systems Medicine and Digestive Disease, Department of Surgery and Cancer, Faculty of
8 Medicine, Imperial College London, Exhibition Road, London SW7 2AZ, UK
9 2Food Microbial Sciences Unit, Department of Food and Nutritional Sciences, School of Chemistry,
10 Food and Pharmacy, Faculty of Life Sciences, The University of Reading, Whiteknights Campus,
11 Reading RG6 6UR, UK
12 3Sorbonne Universities, University Pierre & Marie Curie, University Paris Descartes, Sorbonne Paris
13 Cité, INSERM UMR_S 1138, Cordeliers Research Centre, Paris, France
14 4Sorbonne Paris Cité, Université Denis Diderot, Unité de Biologie Fonctionnelle et Adaptative, CNRS
15 UMR 8251, 75205 Paris, France
16 5Department of Gastroenterology, Guy's and St Thomas' NHS Foundation Trust and King's College
17 London, London, UK
18
19 *These authors made equal contributions to this work; shared first authorship.
20 †Corresponding authors: Anne L. McCartney; Marc-Emmanuel Dumas
21 Keywords: co-metabolic axis; gut–liver axis; metabolomics; Enterobacteriaceae; lactic acid bacteria.
22
23 Email addresses: L. Hoyles, [email protected]; M.L. Jiménez-Pranteda, [email protected]; J.
24 Chilloux, [email protected]; F. Brial, [email protected]; A. Myridakis,
25 [email protected]; T. Aranias, [email protected]; C. Magnan, christophe.magnan@univ-paris-
26 diderot.fr; G.R. Gibson, [email protected]; J.D. Sanderson, [email protected]; J.K.
27 Nicholson, [email protected]; D. Gauguier, [email protected]; A.L. McCartney,
28 [email protected]; M.-E. Dumas, [email protected] 29
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30 ABSTRACT
31 BACKGROUND: The dietary methylamines choline, carnitine and phosphatidylcholine are used by
32 the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However,
33 little is known about the use of trimethylamine N-oxide (TMAO) by this consortium of microbes.
34 RESULTS: A feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial
35 conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to
36 TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated
37 mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture
38 fermentation systems inoculated with human faeces, growth of Enterobacteriaceae was stimulated in
39 the presence of TMAO. Human-derived faecal and caecal bacteria (n = 66 isolates) were screened on
40 solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by 1H-
41 NMR. As with the in vitro fermentation experiments, TMAO stimulated the growth of
42 Enterobacteriaceae; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates
43 of Escherichia coli produced more TMA from TMAO than their faecal counterparts. Lactic acid
44 bacteria produced increased amounts of lactate when grown in the presence of TMAO, but did not
45 produce large amounts of TMA. Clostridia (sensu stricto), bifidobacteria and coriobacteria were
46 significantly correlated with TMA production in the mixed fermentation system but did not produce
47 notable quantities of TMA from TMAO in pure culture.
48 CONCLUSIONS: Reduction of TMAO by the gut microbiota (predominantly Enterobacteriaceae)
49 to TMA followed by host uptake of TMA into the bloodstream from the intestine and its conversion
50 back to TMAO by host hepatic enzymes is an example of metabolic retroconversion. TMAO
51 influences microbial metabolism depending on isolation source and taxon of gut bacterium.
52 Correlation of metabolomic and abundance data from mixed microbiota fermentation systems did not
53 give a true picture of which members of the gut microbiota were responsible for converting TMAO to
54 TMA; only by supplementing the study with pure culture work and additional metabolomics was it
55 possible to increase our understanding of TMAO bioconversions by the human gut microbiota.
56
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57 BACKGROUND
58 Dietary methylamines such as choline, trimethylamine N-oxide (TMAO),
59 phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish,
60 nuts and eggs. It has long been known that gut bacteria are able to use choline in a fermentation-like
61 process, with trimethylamine (TMA), ethanol, acetate and ATP among the known main end-products
62 [1–3]. TMA can be used by members of the order Methanomassiliicoccales (Archaea) present in the
63 human gut to produce methane [4], or taken up by the host. Microbially produced TMA derived from
64 PC is absorbed in the small intestine [5]. TMA diffuses into the bloodstream from the intestine via the
65 hepatic vein to hepatocytes, where it is converted to trimethylamine N-oxide (TMAO) by hepatic
66 flavin-containing mono-oxygenases (FMOs) [6]. The bulk of TMAO, and lesser amounts of TMA,
67 derived from dietary methylamines can be detected in urine within 6 h of ingestion, and both
68 compounds are excreted in urine but not faeces [7,8]. TMAO can also be detected in human skeletal
69 muscle within 6 h of an oral dose of TMAO [8].
70 TMAO present in urine and plasma is considered a biomarker for non-alcoholic fatty liver
71 disease (NAFLD), insulin resistance and cardiovascular disease (CVD) [9–12]. Feeding TMAO to
72 high-fat-fed C57BL/6 mice exacerbates impaired glucose tolerance, though the effect on the gut
73 microbiota is unknown [13]. Low plasma PC and high urinary methylamines (including TMAO) were
74 observed in insulin-resistant mice on a high-fat diet, leading to the suggestion microbial methylamine
75 metabolism directly influences choline bioavailability; however, the microbiota of the mice was not
76 studied nor was labeled choline used to determine the metabolic fate of TMA/TMAO [9]. Choline
77 bioavailability is thought to contribute to hepatic steatosis in patients [11]. Spencer et al. [11]
78 manipulated dietary choline levels in 15 females for 2 weeks and monitored changes in the fecal
79 microbiota during the intervention. They found the level of choline supplementation affected
80 Gammaproteobacteria (inhibited by high levels of dietary choline, negatively correlated with changes
81 in liver fat/spleen fat ratios) and Erysipelotrichi (positively correlated with changes in liver fat/spleen
82 fat ratios), suggesting baseline levels of these taxa may predict the susceptibility of an individual to
83 liver disease. However, the study was under-powered and requires replication with a much larger
84 cohort to fully examine the link between choline bioavailability and liver disease. High levels of
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85 circulating TMAO are associated with CVD [12], potentially via a platelet-mediated mechanism [14].
86 Circulating TMAO has been suggested to play a role in protection from hyperammonemia in mice,
87 acting as an osmoprotectant, and from glutamate neurotoxicity in mice and primary cultures of
88 neurons [15,16]. Recently, chronic exposure to TMAO was shown to attenuate diet-associated
89 impaired glucose tolerance in mice, and reduce endoplasmic reticulum stress and adipogenesis in
90 adipocytes [10]. The relevance of these findings to human health remains to be determined. There is a
91 basal level of TMA and TMAO detected in human urine even in the absence of dietary
92 supplementation [17], suggesting use of (microbial and/or host) cell-derived choline or PC in the
93 intestinal tract by the gut microbiota.
94 While it is well established that choline [2,11,18], PC [2,12] and carnitine [19–22] are used
95 by the human and rodent gut microbiotas to produce TMA, little is known about the reduction of
96 TMAO (predominantly from fish) to TMA (or other compounds) by members of these consortia.
97 TMAO is found at concentrations of 20–120 mg per 100 g fish fillet [23]. Ingestion of fish by humans
98 leads to increased urinary excretion of dimethylamine (DMA) and TMA, from 5.6 to 24.1 and from
99 0.2 to 1.6 µmol/24 h/kg of body weight, respectively [24]. Individuals with trimethylaminuria (fish
100 odour syndrome), in which TMA is not detoxified to TMAO by hepatic FMOs, have been shown to
101 reduce 40–60 % of an oral dose of TMAO to TMA [25]. It was suggested the gut microbiota was
102 responsible for reducing TMAO in these individuals, and in those individuals without
103 trimethylaminuria the TMA was re-oxidized in the liver before being excreted in the urine. The
104 process was termed ‘metabolic retroversion’ “(i.e., a cycle of reductive followed by oxidative
105 reactions to regenerate TMAO)” [25]. The reduction of TMAO to TMA is most commonly associated
106 with the gut microbiota of marine fish, with the TMA generated by these bacteria contributing to the
107 characteristic odour of rotting fish [26]. Members of the class Gammaproteobacteria, which includes
108 a number of food spoilage organisms, are known to reduce TMAO to TMA quantitatively and are
109 unable to reduce TMA further [26,27]. The conversion of TMAO to TMA by bacteria represents a
110 unique form of anaerobic respiration, in which TMAO reductase acts as a terminal electron acceptor
111 (from NADH or formate) for respiratory electron flow [27].
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112 Mining of metagenomic data (torA) suggests Proteobacteria (particularly Escherichia and
113 Klebsiella spp.) are likely to contribute greatest to the production of TMA from TMAO in the human
114 gut via the TMAO reductase pathway, with Actinobacteria (Eggerthellaceae) becoming more
115 important under stress [28]. Other microbial genes are associated with production of TMA from
116 choline (cutC), glycine-betaine (grdH), L-carnitine (cntA) and γ-butyrobetaine (cntA) [4]. A search of
117 the NCBI nucleotide database with the phrase ‘TMAO reductase’ also suggests many other bacteria of
118 human intestinal origin (namely, Salmonella, Helicobacter, Prevotella, Bacillus and Bacteroides spp.)
119 should be able to reduce TMAO to TMA. However, this trait has not been examined in vitro for
120 isolates of intestinal origin.
121 Consequently, we carried out an in vivo study in mice to confirm use of TMAO by the mouse
122 gut microbiota and to allow us to examine metabolic retroconversion of TMAO in a murine model.
123 We then used an in vitro fermentation system to highlight the effect of TMAO on the human faecal
124 microbiota. Finally, we screened a panel of human faecal and caecal/small intestinal isolates to
125 determine which members of the human gut microbiota were able to reduce TMAO to TMA, and
126 whether their metabolism was affected by being grown in the presence of TMAO.
127
128
129 METHODS
130 Animal work
131 Groups of six-week-old C57BL6/J mice (Janvier Labs, Courtaboeuf, France) were received
132 and acclimated in specific pathogen-free (SPF) maintenance conditions. They were fed with a
133 standard chow diet (R04-40, Safe, Augy, France) and were either given free access to tap water or tap
134 water containing the antibiotic cocktail (0.5 g/L vancomycin hydrochloride, 1 g/L neomycin trisulfate,
135 1 g/L metronidazole, 1 g/L ampicillin sodium; all antibiotics were purchased from Sigma-Aldrich) for
-4 136 14 days. Mice were given in the morning by gavage either a solution of d9-TMAO at 1×10 M
137 (Cambridge Isotope Laboratories Inc., DLM-4779-0, UK) or saline and euthanized 6 h later by
138 cervical dislocation. Blood samples were collected by tail tipping every 2 h in Microvette® CB 300
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139 Lithium Heparin (Sarstedt, Marnay, France). Plasma was separated by centrifugation (10 min, 5000 g,
140 4 °C) and stored at -80 °C until analysed by ultra-performance liquid chromatography–tandem mass
141 spectrometry (UPLC–MS/MS).
142
143 UPLC–MS/MS determination of plasma TMA, d9-TMA, TMAO and d9-TMAO
144 UPLC–MS/MS was employed for the determination of TMA, d9-TMA, TMAO and d9-
13 15 145 TMAO. Samples (10 µL) were spiked with 10 µL Internal Standard solution (d9-choline and C3/ N-
146 TMA in water; 1 mg/L, Sigma-Aldrich). Ethyl 2-bromoacetate solution (45 µL) (15 g/L ethyl 2-
147 bromoacetate, 1 % NH4OH in acetonitrile; ChromaSolv grade, Sigma-Aldrich) was added and
13 15 148 derivatization of TMAs (TMA, d9-TMA and C3/ N-TMA) to their ethoxy-analogues was completed
149 after 30 min at room temperature. Protein/lipid precipitation solution (935 µL) (94 % acetonitrile/5
150 %water/1 % formic acid; ChromaSolv grade, Sigma-Aldrich) was added, samples were centrifuged
151 (20 min, 20,000 g, 4 °C) and were transferred to UPLC-autosampler vials. Sample injections (5 µL
152 loop) were performed with a Waters Acquity UPLC-Xevo TQ-S UPLC–MS/MS system equipped
153 with an Acquity BEH HILIC (2.1×100 mm, 1.7 µm) chromatographic column. An isocratic elution
154 was applied with 10 mM ammonium formate (Sigma-Aldrich) in 95:5 (v/v) acetronitrile:water for 7
155 min at 750 µL/min and 50 °C. Positive electrospray (ESI+) was used as ionization source and mass
156 spectrometer parameters were set as follows: capillary, cone and sources voltages at -700, -18 and
157 50 V, respectively, desolvation temperature at 600 °C, desolvation/cone/nebuliser gases were high
158 purity nitrogen (BOC) at 1000 L/h, 150 L/h and 7 bar, respectively. Collision gas was high-purity
159 argon (BOC). Mass spectrometer was operated in multiple reaction monitoring mode. The monitored
160 transitions were the following: for TMA, +146 → +118/59 m/z (23/27 V); for d9-TMA, +155 →
13 15 161 +127/68 m/z (21/23 V); for C3/ N-TMA, +150 → +63/122 m/z (27/22 V); for TMAO, +76 →
162 +59/58 m/z (12/13 V); for d9-TMAO, +85 → +68/66 m/z (18/20 V); and for d9-choline, +108 →
163 +60/45 m/z (20/22 V). The system was controlled by MassLynx software, also used for data
164 acquisition and analysis.
165
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166 1H-NMR spectroscopy and data analysis
167 Medium samples were randomized and centrifuged (5 min, 16000 g). Aliquots (50 µL) of the
168 supernatants were diluted in 550 µL D2O containing 1 mM trimethylsilyl-(2,2,3,3-2H4)-1-propionate.
169 Samples were transferred to 5 mm NMR tubes and measured on a NMR spectrometer (Bruker)
170 operating at 600.22 MHz 1H frequency as described [29]. 1H-NMR spectra were pre-processed and
171 analysed as described [9] using the Statistical Recoupling of Variables-algorithm [30]. Structural
172 assignment was performed as described [31], using in-house and publicly available databases.
173
174 In vitro fermentation systems
175 Freshly voided faecal samples were obtained from three healthy human volunteers (one male
176 and two females; age range 29–31 years), none of whom had been prescribed antibiotics 6 months
177 prior to the study, eaten fish 3 days prior to sample collection or had any history of gastrointestinal
178 disease. The University of Reading’s Research Ethics Committee (UREC) does not require that
179 specific ethical review and approval be given by UREC for the collection of faecal samples from
180 healthy human volunteers to inoculate in vitro fermentation systems. Samples were processed
181 immediately by diluting them 1 in 10 (w/w) in pre-reduced minimal broth and homogenizing them in
182 a filter bag in a stomacher (Stomacher 400 Lab System; Seward) for 2 min at ‘high’ speed. For each
183 donor, a batch culture vessel containing 175 mL of pre-reduced minimal broth was inoculated with
184 20 mL of the faecal homogenate and 5 mL of sterile H2O containing 2 g TMAO dihydrate (Sigma-
185 Aldrich). Control vessels were inoculated with 20 mL of the faecal homogenate and 5 mL of sterile
186 H2O. The final working volume of each batch culture vessel was 200 mL. The pH of each vessel (pH
187 6.5) was controlled automatically by the addition of 2 M HCl or 2 M NaOH. pH controllers were
188 supplied by Electrolab. The contents of each vessel were stirred constantly. An anaerobic
189 environment was maintained by constantly sparging the vessels with O2-free N2. The temperature of
190 each vessel was maintained at 37 °C by use of a circulating waterbath connected to each fermentation
191 vessel. The experiment was run for 9 h, with samples taken at 0, 1, 2, 3, 4, 5, 6 and 9 h.
192
193 Fluorescence in situ hybridization (FISH) analysis
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194 Aliquots (2× 375 µL) of sample were fixed in ice-cold 4 % paraformaldehyde for 4 h, washed
195 in sterile phosphate-buffered saline and stored for FISH analysis as described [32]. Supplementary
196 Table 1 gives details for probes used in this study. Probes were synthesized commercially (MWG-
197 Biotech) and labelled with the fluorescent dye cyanine 3 (Cy3; excitation λ, 514 nm; emission λ, 566
198 nm; fluorescence colour, orange–red). FISH was done as described [32].
199 Slides were viewed under a Nikon E400 Eclipse microscope. DAPI slides were visualized
200 with the aid of a DM400 filter; probe slides were visualized with the aid of a DM575 filter. Cells
201 (between 15 and 50 per field of view) were counted for 15 fields of view, and the numbers of bacteria
202 were determined by using the following equation:
203 DF × ACC × 6,732.42 × 50 × DFsample
204 Where the DF (dilution factor) was calculated by taking into account the concentration of the
205 original sample (375 µL to 300 µL = 0.8×). ACC (average cell count) was determined by counting 15
206 fields of view and assumes that a normal distribution was observed for the counts. The figure
207 6,732.42 refers to the area of the well divided by the area of the field of view. DFsample refers to the
208 dilution of sample used with a particular probe (e.g. 5× for t0 Lab158 counts). The detection limit of
209 this method was 89,766 bacteria/mL of sample (= log10 4.95).
210
211 Screening of bacteria for ability to reduce TMAO
212 Part of an in-house collection (University of Reading) of bacteria isolated from human
213 caecal/small intestinal and faecal samples (Table 1) was screened on minimal agar [g/L: glucose
214 (Fisher Scientific), 4; bacteriological peptone (Oxoid), 20; NaCl (Fisher Scientific), 5; neutral red
215 (Sigma-Aldrich), 0.03; agar technical no. 3 (Oxoid), 15; pH 7] with and without 1 % (w/v) TMAO
216 dihydrate (Sigma-Aldrich). The composition of the agar was based on [27], who used the medium
217 with crystal violet and bile salts to screen members of the Enterobacteriaceae for TMAO reductase
218 activity. Colonies of isolates able to ferment glucose were red, whereas those able to reduce TMAO
219 were white. Growth curves (OD600 measured hourly for 10–12 h, then at 24 h) were determined for
220 selected isolates grown anaerobically at 37 °C in anaerobic minimal broth [g/L: glucose, 4;
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221 bacteriological peptone, 20; NaCl, 5; L-cysteine HCl (Sigma-Aldrich), 0.5; resazurin solution (0.25
222 mg/mL; Sigma-Aldrich), 4 mL; pH 7] with and without 1 % (w/v) TMAO dihydrate. Glucose was
223 substituted by raffinose (Sigma-Aldrich) [33] in the minimal broth when working with bifidobacteria
224 because of the poor growth of these bacteria on the glucose-based medium. pH and metabolite
225 profiles of culture medium were examined at the end of the growth experiment (i.e. at t24).
226
227 Statistical analyses
228 Differences between metabolites produced by faecal and caecal/small intestinal isolates of
229 Escherichia coli and lactic acid bacteria in the presence and absence of TMAO were analysed using
230 Student’s t test. FISH data from the in vitro fermentation systems were analysed using the
231 Kolmogorov–Smirnov test with statistical significance, after correction for multiple testing, taken at P
232 < 0.05. Because of the presence of ties in the metabolite data from in vitro fermentation systems, the
233 bootstrapped Kolmogorov–Smirnov test (10,000 replications) was used with these data. Data from in
234 vitro fermentation systems (FISH and NMR) were correlated using Spearman’s rank correlation
235 (corrected for ties) with results corrected for multiple testing using the method of Benjamini and
236 Hochberg [34].
237
238
239 RESULTS
240 In vivo confirmation of metabolic retroconversion
241 First, to confirm in vivo metabolic retroconversion (i.e. microbial conversion of TMAO to
242 TMA, followed by host conversion of TMA to TMAO), we administered isotopically-labelled d9-
243 TMAO or saline to mice that had or had not been treated with a broad-spectrum antibiotic cocktail for
244 14 days to suppress the gut microbiota [12]. Reduction of d9-TMAO to d9-TMA was quantified in
245 urine by UPLC–MS/MS up to 6 h after d9-TMAO gavage, together with unlabelled TMA and TMAO
246 potentially produced from dietary sources (Fig. 1).
247 In the absence of antibiotics, d9-TMAO was converted to d9-TMA within 2 h of gavage. This
248 conversion was dramatically reduced, leading to a significantly lower concentration of d9-TMA and a
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249 higher concentration of d9-TMAO, when the gut microbiota was suppressed by antibiotics (Fig. 1A–
250 D). We noted that in the absence of antibiotics all animals excreted approximately three times higher
251 levels of urinary unlabelled TMA than TMAO (Fig. 1E–H), corresponding to constitutively low
252 FMO3 activity in mice [35]. Levels of unlabelled TMAO/TMA were not significantly different from
253 one another in the control and d9-TMAO-fed animals, while antibiotic treatment significantly reduced
254 the amount of both TMAO and TMA (Fig. 1E, F). Bioavailability of unlabelled TMAO/TMA, as
255 assessed by area under the curve (AUC), was significantly reduced by antibiotic treatment in both
256 saline and d9-TMAO-gavaged animals, with almost no TMA detected in either experimental group
257 (Fig. 1G, H).
258
259 Effect of TMAO on human gut bacteria within a mixed system
260 After in vivo validation of the role of the gut microbiota in metabolic retroconversion, we
261 analysed the effect of TMAO on the faecal microbiota in an anaerobic batch-culture fermentation
262 system. Fermenter vessels filled with the glucose-containing medium supplemented or not with 1 %
263 (w/v) TMAO were inoculated with faecal slurries from three healthy donors and monitored for 9 h.
264 With the exception of enhanced growth of the Enterobacteriaceae (probe Ent), the presence of
265 TMAO in the medium had no statistically significant effect on the growth of bacteria within the
266 fermentation systems at 9 h (Fig. 2A, Supplementary Figure 1).
267 Huge variability, as measured by 1H-NMR, was observed in the amount of TMA and DMA
268 produced by gut bacteria in the TMAO-containing fermentation systems, with the concentrations of
269 both metabolites increasing steadily from 0 to 9 h and differing significantly (P < 0.05) from the
270 control systems (Fig. 2B). The amount of TMAO in the systems was seen to decrease at 8 h.
271 Correlation of metabolite and FISH data demonstrated Clostridium clusters I and II
272 (Chis150), Enterobacteriaceae (Ent), bifidobacteria (Bif164) and coriobacteriia (Ato291) were
273 associated with TMA, acetate, ethanol and lactate (Fig. 2C). The Betaproteobacteria (Bet42a) were
274 anti-correlated with TMA, acetate, ethanol and lactate, which is unsurprising given this was the only
275 group of bacteria whose representation decreased in the fermentation systems over the course of the
276 experiment (Supplementary Figure 1, Fig. 2C). The Enterobacteriaceae and clostridia were
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277 positively correlated with DMA production. The lactic acid bacteria (Lab158) were not significantly
278 correlated with any of the metabolites in the mixed culture.
279
280 Growth of pure cultures of gut bacteria in the presence of TMAO
281 Using an in-house collection of bacteria, we initially screened isolates on a modified version
282 of the agar of Takahi & Ishimoto [27] as a rapid means of screening bacteria for TMAO reductase
283 activity, and thereby their ability to reduce TMAO to TMA. While members of the
284 Enterobacteriaceae produced expected results (i.e. white colonies when grown in the presence of
285 TMAO and glucose, rather than red colonies on the glucose only control), we observed a number of
286 unexpected outcomes depending on the species under study. For example, colonies of lactic acid
287 bacteria were larger (almost twice their usual size) on TMAO-containing agar than on the glucose
288 control but remained red in colour, suggesting they had not reduced TMAO to TMA at detectable
289 levels but TMAO was influencing their growth. The clostridia examined produced mixed results on
290 the control and TMAO-containing media (i.e. white colonies on both plates or on the control plate
291 only, larger colonies on the TMAO-containing medium but without a colour change of the medium).
292 To determine whether these isolates were converting TMAO to TMA but a low level, we
293 examined the growth of all isolates in liquid culture and metabolites in the spent medium using NMR.
294 The growth of the Enterobacteriaceae was most greatly affected by the presence of TMAO in the
295 medium, with a faster, longer-lasting exponential phase than for the same isolates grown in the
296 control medium (Fig. 3A). pH of the spent medium (after 24 h) when Enterobacteriaceae were grown
297 in the presence of TMAO increased from a mean of 4.7 ± 0.3 (for the control) to 7.6 ± 0.3 (n = 20)
298 (the change in pH is what causes the colonies to appear white on TMAO-containing agar). The
299 growth of lactic acid bacteria, including Enterococcus and Streptococcus (Fig. 3A) spp., was
300 enhanced in the presence of TMAO, but not to the same extent as seen for the Enterobacteriaceae.
301 There was no significant difference (P = 0.27, t test) in the pH of the spent medium for these bacteria
302 after 24 h (mean 4.67 ± 0.9 compared with 4.33 ± 0.27 for the control). The growth of members of
303 Clostridium cluster I (e.g. Clostridium perfringens, Fig. 3A) was not enhanced in the presence of
304 TMAO, though some of these bacteria changed the colour of both the control and TMAO-containing
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305 plates yellow during their growth. The pH of the spent liquid medium confirmed this observation to
306 be due to the alkalinity of the media in the control and TMAO-containing media after 24 h incubation:
307 e.g. Clostridium sporogenes D1(9) (pH 6.25 compared with 6.76 in the control medium), Clostridium
308 paraputrificum L16-FAA6 (pH 6.45 vs 5.38) and Clostridium perfringens L20-BSM1 (pH 5.56 vs
309 4.64).
310 1H-NMR analysis of spent medium from the TMAO-containing and control samples
311 demonstrated that, as expected, the Enterobacteriaceae produced the greatest amount of TMA from
312 TMAO (mean 38.79 ± 11.08 mM compared with 0.03 ± 0.01 mM, n = 20) (Fig. 3B; Supplementary
313 Table 2). Members of the families Peptostreptococcaceae (3.72 mM, n = 1), Clostridiaceae (Cluster
314 I) (2.62 ± 1.83 mM, n = 3), Porphyromonadaceae (1.42 mM, n = 1), Bacteroidaceae (1.40 ± 0.31
315 mM, n = 3), Enterococcaceae (1.19 ± 0.05 mM, n = 5), Erysipelotrichaceae (0.94 mM, n = 1;
316 [Clostridium] ramosum), Staphylococcaceae (0.34 mM, n = 1), Streptococcaceae (0.30 ± 0.16 mM, n
317 = 5), Lactobacillaceae (0.17 ± 0.07 mM, n = 2), Pseudomonadaceae (0.12 mM, n = 1) and
318 Bifidobacteriaceae (0.13 ± 0.1 mM, n = 17) produced low levels of TMA from TMAO (Fig. 3B;
319 Supplementary Table 2). There was great variability in the ability of the bifidobacteria to produce
320 TMA from TMAO, with several isolates and [Clostridium] innocuum, Actinomyces odontolyticus,
321 Fusobacterium ulcerans and Actinomyces viscosus not producing TMA from TMAO (Fig. 3B;
322 Supplementary Table 2).
323
324 Differences in the metabolic capabilities of faecal and caecal Escherichia coli isolates
325 Comparison of the amounts of TMA, and co-metabolites, produced by the faecal (n = 7) and
326 caecal (n = 9) isolates of Escherichia coli demonstrated significantly higher amounts of TMA were
327 produced by the caecal isolates compared with the faecal isolates in the TMAO-containing medium
328 (Fig. 3C). Escherichia coli of caecal origin produced more TMA than faecal isolates of the same
329 bacterium or other enterobacteria (Hafnia, Citrobacter and Klebsiella spp.) (Supplementary Table
330 2). The faecal isolates produced more acetate and lactate than the caecal isolates when grown in the
331 control medium. Taken together, these results demonstrate the different metabolic capabilities of
332 isolates of Escherichia coli recovered from different regions of the human gut.
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333
334 Lactic acid bacteria produce more lactate in the presence of TMAO
335 Differences were also seen in the amount of lactic acid produced by lactic acid bacteria in the
336 presence and absence of TMAO (Fig. 3B, D). In raffinose-containing medium, bifidobacteria
337 produced increased of amounts of lactate when grown in the presence of TMAO (the bifidobacteria
338 grew poorly, if at all, in glucose-containing media). Unlike the bifidobacteria, the Streptococcaceae
339 and Enterococcaceae grew well in the glucose-containing medium and produced over 25 mM lactic
340 acid in the TMAO-containing samples compared with <5 mM in the control samples (Fig. 3D). To the
341 best of our knowledge, this is the first time TMAO has been shown to influence the metabolism of gut
342 bacteria – specifically lactic acid bacteria – without producing appreciable amounts of TMA.
343
344
345 DISCUSSION
346 TMAO is a circulating metabolite produced as a direct result of microbial degradation of
347 dietary methylamines in the intestinal tract, and can be readily detected along with its precursor TMA
348 in human urine, blood and skeletal muscle [8,12,18,22]. It worsens atherosclerosis in some mouse
349 models of CVD, and is positively correlated with CVD severity in humans. Beneficial effects
350 associated with TMAO include potential protection from hyperammonia and glutamate neurotoxicity,
351 alleviation of endoplasmic reticulum stress and improved glucose homeostasis by stimulating insulin
352 secretion by pancreatic cells [10,15,16].
353 Over many decades it has been established that choline, PC and carnitine are dietary
354 methylamines that contribute directly to microbiome-associated circulating levels of TMAO found in
355 humans and other animals – e.g. [2,12,18,19,21,22]. However, TMAO itself is a water-soluble
356 osmolyte found in high abundance in fish. Based on their observations that individuals with
357 trimethylaminuria could reduce an oral dose of TMAO to TMA could not detoxify it to TMAO, al-
358 Waiz et al. [25] suggested the gut microbiota could use TMAO as a substrate, and in those individuals
359 without trimethylaminuria the TMA was re-oxidized in the liver before being excreted in the urine.
360 This led these authors to propose the process of ‘metabolic retroversion’. Reference to the literature
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361 associated with fish spoilage and recent mining of metagenomic data have predicted members of the
362 Enterobacteriaceae (particularly Escherichia coli and Klebsiella pneumoniae) have the potential to
363 convert TMAO to TMA in the intestinal tract, but this has not been tested in vitro or in vivo to date
364 [4,8,26–28]. Consequently, we instigated this study to demonstrate metabolic retroconversion of
365 TMAO, and to determine the effect of TMAO on the growth and metabolism of human-derived
366 intestinal bacteria in pure and mixed cultures.
367 Through in vivo administration of deuterated TMAO to mice via oral gavage in mice, we
368 unambiguously demonstrated that TMAO is converted to TMA, with this TMA detectable in plasma
369 within 2 h of administration. This conversion was highly dependent on the gut microbiota, as
370 conversion of TMAO to TMA was dramatically reduced when the microbiota was suppressed by
371 treatment of animals with broad-spectrum antibiotics. Even in the presence of antibiotics, there was
372 low-level conversion of TMAO to TMA, suggesting a subpopulation of the microbiota was resistant
373 to the antibiotics used in our experiment. However, administration of a broad-spectrum antibiotic
374 cocktail for 14 days has been demonstrated to be an effective means of suppressing the gut microbiota
375 in studies associated with gut microbial use of dietary methylamines [12].
376 Our in vivo experiment clearly demonstrates the gut microbiota converts TMAO to TMA, and
377 that this TMA is re-oxidized to TMAO, in line with the process of ‘metabolic retroversion’ [25]. Gut-
378 associated microbial conversion of the majority of TMAO to TMA is at odds with recent findings [8],
379 in which it was suggested TMAO is taken up intact and not metabolised by the gut microbiota of
380 humans. The ratio of TMAO-to-TMA is around 10:1 in humans and 1:10 in mice, meaning TMAO
381 was N-oxidized before Taesuwan et al. [8] were able to observe it in circulating blood of their human
382 subjects. In a human system with high FMO3 activity, 17O-labelled TMAO would need to be used in
383 any study evaluating O turnover to allow calculation of the true rate of retroconversion.
384 Based on work done in mice [10,36], metabolic retroconversion of TMAO may be protective,
385 and may even go some way to explaining why TMA and TMAO are detected at low levels in urine in
386 the absence of dietary methylamines [17]. Chronic exposure of high-fat-fed mice to TMAO reduced
387 diet-associated endoplasmic stress and adipogenesis, and improved glucose tolerance [10]. Exposure
388 of high-fat-fed mice to TMA reduced low-grade inflammation and insulin resistance via inhibition of
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389 interleukin-1 receptor-associated kinase 4 (IRAK-4) [36]. Concentrations of the microbial signalling
390 metabolite TMA used by Chilloux et al. [36] were comparable to those found in normal human
391 plasma. Continual exposure of the human system to TMA/TMAO may bring similar benefits to host
392 metabolic and inflammatory responses; these remain to be studied.
393 Having conducted our in vivo experiment in mice, we examined the ability of a range of
394 human-derived gut bacteria to convert TMAO to TMA. In the mixed microbiota system and in pure
395 cultures, the growth of the Enterobacteriaceae – the main TMA-producers – was quickly affected by
396 the presence of TMAO. This is likely to happen in the human gut also. Consequently, we believe
397 dietary TMAO undergoes metabolic retroconversion in mammals, with the TMA produced as a result
398 of bacterial activity in the gut available to the host for conversion back to TMAO by FMOs in
399 hepatocytes. It should be noted that [8] did not suppress or monitor the intestinal/faecal microbiota
400 when they administered isotopically labeled TMAO to humans, nor did they measure d9-TMA and d9-
401 TMAO in the portal vein, bypassing subsequent hepatic N-oxidation of d9-TMA, so it is not possible
402 to interpret their results in the context of presence/absence of microbial activity.
403 Of note is the finding that caecal isolates of enterobacteria produce more TMA from TMAO
404 than faecal isolates of the same bacterium. The speed with which TMAO was reduced to TMA by the
405 Enterobacteriaceae in the present study suggests bacterial conversion of TMAO to TMA takes place
406 in the small intestine/proximal colon of humans and small intestine/caecum of mice. It is, therefore,
407 unsurprising that caecal bacteria – representing the microbiota present at the intersection of the small
408 and large intestine – are metabolically more active than their faecal counterparts with respect to
409 TMAO metabolism. This finding is relevant to functional studies of the gut microbiota where gene
410 annotations are based largely on faecal isolates, whose functionalities may be greatly different from
411 those of bacteria in other regions of the human intestinal tract [37,38]. It has already been
412 demonstrated that the microbiota of the small intestine is enriched for functions associated with rapid
413 uptake and fermentation of simple carbohydrates compared with the faecal microbiota, and that
414 streptococci isolated from this niche are functionally very different from the same bacteria isolated
415 from different habitats [37,38]. It is, therefore, important we characterize the functions and genomes
416 of bacteria isolated from all regions of the intestinal tract, not just those of faecal bacteria, to gain a
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417 true picture of how microbial activity influences host health.
418 Enterobacteriaceae made the greatest contribution to the conversion of TMAO to TMA, both
419 in pure culture and in a mixed microbiota. These Gram-negative bacteria are a source of the virulence
420 factor lipopolysaccharide (LPS), which is associated with low-grade inflammation in high-fat-fed
421 mice and elevated plasma levels that define metabolic endotoxaemia [39,40]. High-fat feeding has
422 been shown to increase the representation of Enterobacteriaceae in the caecal microbiota of obesity-
423 prone Sprague–Dawley rats [41], though this mode of feeding is known to modulate the microbiota of
424 mice independent of obesity [42]. Non-LPS-associated virulence of Enterobacteriaceae, Vibrio
425 cholerae and Helicobacter pylori is increased when these bacteria are grown anaerobically or
426 microaerophilically in the presence of as little as 5–10 mM TMAO [43–46], and may be an additional
427 means by which the gut microbiota contributes to CVD and other diseases in which increased
428 representation of Gammaproteobacteria is observed. This warrants attention in future animal studies.
429 Lactic acid bacteria clearly grow better in the presence of TMAO. The relatively high
430 concentration (1 %) of TMAO used in this study may have contributed to this improved growth, as
431 TMAO is an osmolyte that stabilizes proteins. Future work will involve growing lactic acid bacteria in
432 a range of TMAO concentrations to determine how this compound affects their growth and gene
433 expression, and comparing faecal and caecal isolates. Similar to the Enterobacteriaceae, a large
434 number of these bacteria are facultative anaerobes able to grow over a range of conditions, and whose
435 representation is increased in obese, and cirrhotic patients [47,48]. Streptococcus and Enterococcus
436 spp. are commensal lactic acid bacteria of the gut microbiota known to modulate immune function;
437 but little is known about their metabolic activities in mixed microbial populations [49]. Understanding
438 how commensal lactic acid bacteria influence the host in dysbiosis in mixed microbial communities
439 may allow the development of approaches to modulate their activity and influence host health.
440 With respect to the lactic acid bacteria, it is important to note that our mixed culture work did
441 not highlight these as being relevant to TMAO metabolism. This is unsurprising given these bacteria
442 do not produce large quantities of TMA from TMAO. However, we have shown their metabolism is
443 affected by presence of TMAO in growth medium, and the increased lactate they produce in its
444 presence may contribute to cross-feeding associated with short-chain fatty acid production [50]. It is
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445 difficult to determine relevance of correlations from mixed microbial ecosystems in the absence of
446 isotope labelling or pure culture work: i.e. correlation does not equate with causation. As an example,
447 three (Clostridium clusters I and II, bifidobacteria, coriobacteriia) of the four groups of bacteria
448 correlated with TMA production in our fermentation study did not produce notable quantities of TMA
449 from TMAO based on our pure culture work. Therefore, correlating microbiota and metabolite data
450 derived from complex systems will not give a true picture of which members of microbiota contribute
451 to specific metabolic processes, and work with pure cultures is required to supplement functional
452 studies to increase our understanding of poorly understood microbially driven metabolic processes
453 within the human gut.
454
455
456 CONCLUSIONS
457 We have demonstrated metabolic retroconversion – another example of host–microbial co-
458 metabolism – occurs in the mammalian system with respect to TMAO, whereby TMAO is reduced by
459 the gut microbiota to TMA and regenerated by host hepatic enzymes. We have also demonstrated that
460 growth and metabolism of members of the gut microbiota are affected by TMAO in a source- and
461 taxon-dependent manner, with the family Enterobacteriaceae making the greatest contribution to
462 production of TMA in the gut.
463
464
465 ABBREVIATIONS
466 AUC, area under the curve; DMA, dimethylamine; FISH, fluorescence in situ hybridization; FMO,
467 flavin mono-oxygenase; MMA, monomethylamine; PC, phosphatidylcholine; TMA, trimethylamine;
468 TMAO, trimethylamine N-oxide; UPLC–MS/MS, ultra-performance liquid chromatography–tandem
469 mass spectrometry.
470
471
472 DECLARATIONS
17 bioRxiv preprint doi: https://doi.org/10.1101/225581; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
473 Ethics approval: All animal procedures were authorized following review by the institutional ethics
474 committee (Sorbonne Universities) and carried out under national license conditions. Ethical approval
475 to collect caecal effluent from patients was obtained from St Thomas’ Hospital Research Ethics
476 Committee (06/Q0702/74) covering Guy’s and St Thomas’ Hospitals, and transferred by agreement to
477 London Bridge Hospital. Patients provided written consent to provide samples.
478 Consent for publication: Not applicable.
479 Availability of data and material: The datasets used and/or analysed during the current study are
480 available from the corresponding authors upon reasonable request.
481 Competing interests: The authors have no competing interests.
482 Funding: MLJP was sponsored by Fundación Alfonso Martin Escudero (Spain). Work was supported
483 by a grant from the EU-FP7 (METACARDIS, HEALTH-F4-2012-305312) to DG and MED. LH is in
484 receipt of an MRC Intermediate Research Fellowship in Data Science (grant number MR/L01632X/1,
485 UK Med-Bio).
486 Authors’ contributions: MLJP, LH and ALM performed the microbiological work, JC was responsible
487 for NMR analysis and interpretation of the NMR data, TA and FB did the animal work, MED
488 supervised the NMR work, ALM supervised the microbiological work, CM provided invaluable
489 advice for the labelling study and DG supervised the animal work. All authors contributed to the
490 writing of the manuscript.
491 Acknowledgements: Not applicable.
492
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608 [45] Paiva JB, Penha Filho RA, Pereira EA, Lemos, MV, Barrow PA, Lovell MA, et al. The 609 contribution of genes required for anaerobic respiration to the virulence of Salmonella enterica 610 serovar Gallinarum for chickens. Braz J Microbiol. 2009;40:994–1001. 611 [46] Wu D, Cao M, Peng J, Li N, Yi S, Song L, et al. The effect of trimethylamine N-oxide on 612 Helicobacter pylori-induced changes of immunoinflammatory genes expression in gastric 613 epithelial cells. Int Immunopharmacol. 2017;43:172-8. 614 [47] Qin N, Yang F, Li A, Prifti E, Chen Y, Shao L, et al. Alterations of the human gut microbiome 615 in liver cirrhosis. Nature. 2014;513:59-64. 616 [48] Le Chatelier E, Nielsen T, Qin J, Prifti E, Hildebrand F, Falony G, et al. Richness of human gut 617 microbiome correlates with metabolic markers. Nature. 2013;500:541-6. 618 [49] van den Bogert B, Meijerink M, Zoetendal EG, Wells JM, Kleerebezem M. Immunomodulatory 619 properties of Streptococcus and Veillonella isolates from the human small intestine microbiota. 620 PLoS One. 2014;9:e114277. 621 [50] Hoyles L, Wallace RJ. Gastrointestinal tract: intestinal fatty acid metabolism and implications 622 for health. In Handbook of Hydrocarbon and Lipid Microbiology, vol. 4, part 7, Human-Animal- 623 Plant Health and Physiology Consequences of Microbial Interactions with Hydrocarbons and 624 Lipids. 2010, 3120-32. Edited by T. McGenity, K. N. Timmis, J. R. van der Meer & V. de 625 Lorenzo. New York: Springer. 626 [51] Roger LC, Costabile A, Holland DT, Hoyles L, McCartney AL. Examination of faecal 627 Bifidobacterium populations in breast- and formula-fed infants during the first 18 months of life. 628 Microbiology. 2010;156:3329-41. 629 [52] Hoyles L, Murphy J, Neve H, Heller KJ, Turton JF, Mahony J, et al. Klebsiella pneumoniae 630 subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman. 631 PeerJ. 2015;doi:10.7717/peerj.1061. 632
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633 FIGURE LEGENDS
634 Fig. 1. In vivo confirmation of metabolic retroconversion of TMAO. Reduction of d9-TMAO to d9-
635 TMA was quantified by UPLC–MS/MS up to 6 h after d9-TMAO gavage and antibiotic treatment,
636 together with unlabelled TMA and TMAO levels. Plasma quantification of post-gavage (A) d9-TMA
637 and (B) d9-TMAO. *, Significantly (P < 0.05; t test and corrected for multiple comparison using the
638 Holm–Sidak method) different from the respective groups not treated with antibiotics. (C) d9-TMA
639 bioavailability (AUC). (D) d9-TMAO bioavailability (AUC). Plasma quantification of post-gavage
640 unlabelled/endogenous (E) TMA and (F) TMAO. *, Significant between d9 and d9 antibiotic
641 treatment; $, significant between TMAO and TMAO antibiotic treatment. (G) Unlabelled/endogenous
642 TMA bioavailability (AUC). (H) Unlabelled/endogenous TMAO bioavailability (AUC). Data (n = 6
643 per group) are shown as mean ± SEM. (A, B, E and F). Differences between the bioavailabilities (C,
644 D, G and H) were assessed using one-way analysis of variance (ANOVA), followed by Holm–Sidak
645 post hoc tests. Data with different superscript letters are significantly different (P < 0.05).
646
647 Fig. 2. Effect of TMAO on mixed faecal microbial population in vitro. (A) Enumeration of selected
648 bacteria in fermentation vessels by FISH analysis. Red lines, TMAO-containing systems; blue lines,
649 negative controls. Data are shown as mean + SD (n = 3). Eub338, total bacteria; Ent,
650 Enterobacteriaceae; Bif164, Bifidobacterium spp.; Lab158, lactic acid bacteria. *, Statistically
651 significantly different (adjusted P < 0.05) from the control at the same time point. Full data are shown
1 652 in Supplementary Figure 1. (B) H-NMR data for batch culture samples. Data are shown mean ± SD
653 (n = 3). Red lines, TMAO-containing systems; blue lines, negative controls. *, Statistically
654 significantly different (P < 0.05) from the negative control at the same time point. (C) Bidirectional
655 clustering of correlation matrix of FISH data and data for the six metabolites found in highest
656 amounts in the NMR spectra from the batch-culture samples. +, Adjusted P value (Benjamini–
657 Hochberg) statistically significant (P < 0.05). FISH and metabolite data and a table of correlations and
658 adjusted P values (Benjamini–Hochberg) for the batch-culture samples are available in
659 Supplementary Tables 3–5.
660
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661 Fig. 3. Influence of TMAO on growth and metabolism of pure cultures of gut bacteria. (A)
662 Representative growth curves for isolates grown in the presence and absence of TMAO. Red lines,
663 TMAO-supplemented cultures; blue lines, negative controls. Data are shown as mean ± SD (n = 3).
664 (B) Biplot showing production of various metabolites when isolates were grown in the presence of
665 TMAO. Summary of data from Supplementary Table 2. The larger a circle, the more of the
666 metabolite produced by an isolate. (C) Differences in metabolites produced when caecal and faecal
667 isolates of Escherichia coli were grown in the presence (+) and absence (–) of 1 % TMAO. Adjusted
668 (Benjamini–Hochberg) P values indicate the caecal isolates were significantly different from the
669 faecal isolates for a particular metabolite. (D) Lactate production by lactic acid bacteria was increased
670 in the presence of TMAO. Enterobacteriaceae, n = 20; Bifidobacteriaceae, n = 17; Streptococcaceae,
671 n = 7; Enterococcaceae, n = 5. Members of the Enterococcaceae and Streptococcaceae are
672 homofermenters (produce only lactic acid from glucose fermentation), whereas the
673 Bifidobacteriaceae are heterofermenters (produce ethanol, CO2 and lactic acid from glucose
674 fermentation), though it should be noted the bifidobacteria included in this study were grown on
675 raffinose-containing media. Red, TMAO-containing medium; blue, negative control. *, Statistically
676 significantly different from its negative control (adjusted P value < 0.05).
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677 Fig. 1
678
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679 Fig. 2
680
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681 Fig. 3
682
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683 Table 1. Details for human-derived gut bacteria screened for their ability to reduce or utilize TMAO
Isolate* Identified as Source Reference Facultative anaerobe? D2(9) [Clostridium] innocuum Human faeces M.L. Jiménez-Pranteda, unpublished No L16-FAA1 [Clostridium] ramosum Human caecum L. Hoyles, unpublished No L12-BSM1 Actinomyces odontolyticus Human caecum L. Hoyles, unpublished No L6-BSM10 Actinomyces viscosus Human caecum L. Hoyles, unpublished No L6-FAA7 Bacteroides fragilis Human caecum L. Hoyles, unpublished No D1(4) Bacteroides vulgatus Human faeces M.L. Jiménez-Pranteda, unpublished No L9-FAA7 Bacteroides vulgatus Human caecum L. Hoyles, unpublished No DSM 20083T Bifidobacterium adolescentis Human intestine DSMZ† No LCR26 Bifidobacterium animalis subsp. lactis Infant faeces [51] No LCR11 Bifidobacterium bidifum Infant faeces [51] No L25-MRS2 Bifidobacterium bifidum Human caecum L. Hoyles, unpublished No LCR5 Bifidobacterium breve Infant faeces [51] No LCR8 Bifidobacterium breve Infant faeces [51] No LCR1 Bifidobacterium dentium Infant faeces [51] No DSM 20093T Bifidobacterium gallicum Human intestine DSMZ No LCR6 Bifidobacterium longum Infant faeces [51] No DSM 20088T Bifidobacterium longum subsp. infantis Infant intestine DSMZ No LCR2 Bifidobacterium longum subsp. infantis Infant faeces [51] No DSM 20219T Bifidobacterium longum subsp. longum Human intestine DSMZ No LCR3 Bifidobacterium pseudocatenulatum Infant faeces [51] No L19-MRS1 Bifidobacteroum longum subsp. longum Human caecum L. Hoyles, unpublished No L25-MRS8 Bifidobacteroum longum subsp. longum Human caecum L. Hoyles, unpublished No L26-MRS4 Bifidobactrium animalis subsp. lactis Human caecum L. Hoyles, unpublished No L26-FAA1 Citrobacter gillenii Human caecum L. Hoyles, unpublished Yes L8-FAA3 Citrobacter koseri Human caecum L. Hoyles, unpublished Yes L16-FAA6 Clostridium paraputrificum Human caecum L. Hoyles, unpublished No L20-BSM1 Clostridium perfringens Human caecum L. Hoyles, unpublished No D1(9) Clostridium sporogenes Human faeces M.L. Jiménez-Pranteda, unpublished No D2(14) Enterococcus faecalis Human faeces M.L. Jiménez-Pranteda, unpublished Yes D3(1) Enterococcus faecalis Human faeces M.L. Jiménez-Pranteda, unpublished Yes D5(2) Enterococcus faecalis Human faeces M.L. Jiménez-Pranteda, unpublished Yes D6(1) Enterococcus faecium Human faeces M.L. Jiménez-Pranteda, unpublished Yes D6(5) Enterococcus gallinarum Human faeces M.L. Jiménez-Pranteda, unpublished Yes D1(2) Escherichia coli Human faeces M.L. Jiménez-Pranteda, unpublished Yes D2(1) Escherichia coli Human faeces M.L. Jiménez-Pranteda, unpublished Yes D2(2) Escherichia coli Human faeces M.L. Jiménez-Pranteda, unpublished Yes D2(8) Escherichia coli Human faeces M.L. Jiménez-Pranteda, unpublished Yes D3(8) Escherichia coli Human faeces M.L. Jiménez-Pranteda, unpublished Yes D4(15) Escherichia coli Human faeces M.L. Jiménez-Pranteda, unpublished Yes D5(1) Escherichia coli Human faeces M.L. Jiménez-Pranteda, unpublished Yes L1-FAA5 Escherichia coli Human caecum L. Hoyles, unpublished Yes L13-FAA2 Escherichia coli Human caecum L. Hoyles, unpublished Yes L16-FAA5 Escherichia coli Human caecum L. Hoyles, unpublished Yes L19-FAA2 Escherichia coli Human caecum L. Hoyles, unpublished Yes L20-FAA3 Escherichia coli Human caecum L. Hoyles, unpublished Yes L24-FAA5 Escherichia coli Human caecum L. Hoyles, unpublished Yes L5-FAA2 Escherichia coli Human caecum L. Hoyles, unpublished Yes L6-FAA1 Escherichia coli Human caecum L. Hoyles, unpublished Yes L9-MRS1 Escherichia coli Human caecum L. Hoyles, unpublished Yes L9-FAA5 Fusobacterium ulcerans Human caecum L. Hoyles, unpublished Yes L15-FAA9 Hafnia paralvei Human caecum L. Hoyles, unpublished No L4-FAA5 Klebsiella pneumoniae subsp. pneumoniae Human caecum [52] Yes L26-MRS5 Lactobacillus fermentum Human caecum L. Hoyles, unpublished No
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L26-FAA6 Lactobacillus rhamnosus Human caecum L. Hoyles, unpublished No L13-FAA10 Parabacteroides johnsonii Human caecum L. Hoyles, unpublished No D4(1) Paraclostridium bifermentans Human faeces M.L. Jiménez-Pranteda, unpublished No L1-FAA6 Pseudomonas aeruginosa Human caecum L. Hoyles, unpublished Yes D2(4) Staphyloccoccus hominis Human faeces M.L. Jiménez-Pranteda, unpublished Yes D1(5) Streptococcus anginosus Human faeces M.L. Jiménez-Pranteda, unpublished Yes D5(12) Streptococcus anginosus Human faeces M.L. Jiménez-Pranteda, unpublished Yes L25-MRS1 Streptococcus gallolyticus Human caecum L. Hoyles, unpublished Yes L4-MRS5 Streptococcus oralis Human caecum L. Hoyles, unpublished Yes L4-MRS1 Streptococcus sanguinis Human caecum L. Hoyles, unpublished Yes D4(3) Streptococcus sp. Human faeces M.L. Jiménez-Pranteda, unpublished Yes L26-MRS7 Streptococcus vestibularis Human caecum L. Hoyles, unpublished Yes LCR4 Unknown Bifidobacterium Infant faeces [51] No 684
685 *Ln- prefix, different numbers indicate isolates recovered from different individuals.
686 †DSMZ, Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures.
29